The human pathogen Bordetella pertussis causes the respiratory infection know as whooping cough (pertussis). Today the disease still remains a major pulmonary health problem nationally and internationally. Pertussis predominately occurs in young children, yet it can also attack people of all ages. Our knowledge of how B. pertussis regulates virulence gene expression is still incomplete. This research grant proposes to investigate the role of two previously unidentified B. pertussis transcriptional regulators, Btr (Bordetella transcriptional regulator) and Baf (Bvg accessory factor), in the control of Bordetella metabolism and pathogenesis. These studies should expand our understanding of gene regulation in B. pertussis. It will likely lead to a better understanding of the physiology of this pathogen, to the identification of new virulence factors, and to a more complete understanding of how this microbe causes disease. The objectives of the research are: 1) To identify and isolate genes of B. pertussis whose transcription is controlled by Btr. A recently developed Btr titration assay will be used to identify Btr- regulated genes from a B. pertussis genomic plasmid library. Btr- regulated genes will be characterized and selected genes will be sequenced. 2) To determine whether baf expression is regulated by the BvgAS two-component regulatory system and to define the cis-acting elements involved in baf expression. First, baf expression in wildtype and bvg B. pertussis strains grown in the presence or absence of modulating agents (e.g., MgSO4) will be examined by Northern analysis and S1 nuclease protection assays. Second, the 5'-upstream region of baf expression and regulation will be identified by analysis of chromosomal baf promoter-lacZ transcriptional fusions in B. pertussis. 3) To determine how Baf, BvgA, and BvgS interact with each other and with the promoter regions of the ptx and cya genes. To accomplish this; Baf, BvgA, and BvgS proteins will be overexpressed and purified. Interactions of these regulatory proteins with promoter regions will be analyzed via gel retardation and DNase I protection analysis. 4) To determine the role Btr, Btr-regulated genes, and Baf play in the virulence of Bordetella pertussis. Mutations will be constructed in these genes which will then be introduced into the chromosome of virulent B. pertussis by gene replacement techniques. The Btr-regulated gene mutants will be analyzed for growth characteristics and, along with the Baf-deficient mutant, will be analyzed for levels of virulence factor production. The persistence and lethality of these mutants will be tested in the mouse intranasal infection model.